DNA Technology CHAPTER 12. The DNA of two people of the same sex is 99.9% identical

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1 CHAPTER 12 DNA Technology Figures PowerPoint Lecture Slides for Essential Biology, Second Edition & Essential Biology with Physiology Presentation prepared by Chris C. Romero Neil Campbell, Jane Reece, and Eric Simon The DNA of two people of the same sex is 99.9% identical Animals, plants, and even bacteria can be genetically modified to produce human proteins The first use of DNA fingerprinting in a murder case proved one man innocent and another guilty Genetically modified strains account for half of the U.S. corn crop 1

2 BIOLOGY AND SOCIETY: HUNTING FOR GENES DNA technology is a set of methods for studying and manipulating genetic material These techniques have brought about many remarkable scientific advances Genetically modified food DNA fingerprinting The Human Genome Project The goal of the Human Genome Project is to determine the nucleotide sequence of all DNA in the human genome Hundreds of diseaseassociated genes have already been identified Example: Parkinson disease Figure 12.1 RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology is a set of techniques for combining genes from different sources into a single DNA molecule An organism that carries recombinant DNA is called a genetically modified (GM) organism Recombinant DNA technology is applied in the field of biotechnology Biotechnology uses various organisms to perform practical tasks 2

3 Recombinant DNA (host DNA plus gene of interest) Host cell 2 DNA Cell multiplies and produces protein 1 Cell containing gene of interest Gene of interest 3 Genes may be inserted into another organism Protein may be harvested OR Gene for pest resistance inserted into plants Protein dissolves blood clots in heart attack therapy Figure 12.2 From Humulin to Genetically Modified Foods By transferring the gene for a desired protein product into a bacterium, proteins can be produced in large quantities Making Humulin In 1982, the world s first genetically engineered pharmaceutical product was produced Humulin, human insulin, was produced by genetically modified bacteria Figure

4 Prior to the development of Humulin, diabetes was treated using insulin from cows and pigs These types of insulin can cause adverse reactions in recipients Humulin was the first recombinant DNA drug approved by the FDA DNA technology is also helping medical researchers develop vaccines A vaccine is a harmless variant or derivative of a pathogen Vaccines are used to prevent infectious diseases Figure 12.4 Genetically Modified (GM) Foods Today, DNA technology is quickly replacing traditional plant-breeding programs In 2002, roughly half of the American crops of soybeans and corn were genetically modified in some way Figure 12.5a 4

5 Corn has been genetically modified to resist insect infestation This corn has been damaged by the European corn borer Figure 12.5b Golden rice has been genetically modified to contain beta-carotene Our bodies use betacarotene to make vitamin A Figure 12.6 Farm Animals and Pharm Animals While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase These transgenic sheep carry a gene for a human blood protein This protein may help in the treatment of cystic fibrosis Figure

6 While transgenic animals are currently used to produce potentially useful proteins, none are yet found in our food supply It is possible that DNA technology will eventually replace traditional animal breeding Recombinant DNA Techniques Bacteria are the workhorses of modern biotechnology To work with genes in the laboratory, biologists often use bacterial plasmids Plasmids are small, circular DNA molecules that are separate from the much larger bacterial chromosome Plasmids Bacterial chromosome Remnant of bacterium Figure

7 Plasmids can easily incorporate foreign DNA Plasmids are readily taken up by bacterial cells Plasmids then act as vectors, DNA carriers that move genes from one cell to another Recombinant DNA techniques can help biologists produce large quantities of a desired protein Bacterial cell Plasmid 1 Isolate DNA from two sources 2 Gene V Cut both DNAs Gene V 3 4 Other genes Recombinant DNA plasmids Recombinant bacteria Bacterial clones Human cell DNA Mix the DNAs and join them together Bacteria take up recombinant plasmids 5 Clone the bacteria DNA fragments 6 Find the clone with gene V 7 Grow bacteria and isolate protein V Protein V Figure 12.9 A Closer Look: Cutting and Pasting DNA with Restriction Enzymes Recombinant DNA is produced by combining two ingredients A bacterial plasmid The gene of interest To combine these ingredients, a piece of DNA must be pasted into a plasmid 7

8 This pasting process can be accomplished using restriction enzymes These enzymes cut DNA at specific nucleotide sequences The places where DNA is cut are called restriction sites Many of these restriction sites leave staggered cuts that yield two double-stranded DNA fragments with single-stranded ends called sticky ends These are the key to joining DNA restriction fragments Recognition sequence for a restriction enzyme 1 Restriction enzyme cuts DNA into fragments Sticky end 2 Addition of a DNA fragment from another source Sticky end 3 Fragments stick together by base pairing 4 DNA ligase joins the strands Recombinant DNA molecule Figure A Closer Look: Obtaining the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? The shotgun approach is one way to synthesize a gene of interest Millions of recombinant plasmids containing different segments of foreign DNA are produced This collection is called a genomic library 8

9 Once a genomic library is created, biologists must identify the bacterial clone containing the desired gene A specific sequence of radioactive nucleotides matching those in the desired gene can be created This type of labeled nucleic acid molecule is called a nucleic acid probe How a DNA probe tags a gene Radioactive probe (DNA) Mix with single-stranded DNA from various bacterial clones Single-stranded DNA Base pairing indicates the gene of interest Figure Reverse transcriptase is another method for synthesizing a gene of interest Cell nucleus Exon Intron Exon Intron Exon DNA of eukaryotic gene 1 Transcription RNA transcript 2 Introns removed and exons spliced together mrna Cytoplasm Test tube Reverse transcriptase 3 4 Isolation of mrna from cell and addition of reverse transcriptase Synthesis of cdna strand Figure cdna strand being synthesized cdna of gene without introns 5 Synthesis of second DNA strand 9

10 DNA FINGERPRINTING AND FORENSIC SCIENCE DNA technology has rapidly revolutionized the field of forensics Forensics is the scientific analysis of evidence from crime scenes DNA fingerprinting can be used to determine whether or not two samples of genetic material are from the same individual Crime scene Suspect 1 Suspect 2 1 DNA collected 2 DNA amplified if necessary 3 DNA cut into fragments 4 DNA fragments compared Figure Murder, Paternity, and Ancient DNA The First Case In 1983 and again in 1986, young girls were raped and murdered near Narborough, England The killer left behind few clues, except for semen A man confessed to the second murder, but denied committing the first 10

11 Police turned to a professor at Leicester University who had recently developed the first DNA fingerprint identification system He compared DNA from samples collected at both murder scenes and concluded that both murders had been committed by the same killer Surprisingly, DNA from the suspect did not match either crime scene The case was finally broken using DNA fingerprinting and the killer was brought to justice Crimes and Other Investigations Since its introduction in 1986, DNA fingerprinting has become a standard part of law enforcement This type of evidence has been used in many cases It can prove innocence or guilt Figure DNA fingerprinting has also been used to identify victims of the World Trade Center attack In evolution research, this technique has been used to study ancient pieces of DNA, such as that of Cheddar Man 11

12 DNA Fingerprinting Techniques The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) is a technique by which any segment of DNA can be amplified (cloned) Through PCR, scientists can obtain enough DNA from even minute amounts of blood or other tissue to allow DNA fingerprinting DNA amplification by PCR Initial DNA segment 1 2 Number of DNA molecules 4 8 Figure Restriction Fragment Length Polymorphism (RFLP) Analysis DNA fingerprinting relies on indirect methods to compare samples One method is called RFLP analysis RFLP analysis is the comparison of a set of restriction fragments produced by DNA from different individuals RFLP stands for restriction fragment length polymorphism 12

13 The goal of DNA fingerprinting by RFLP analysis is to determine whether or not samples of DNA contain identical markers A genetic marker is a chromosomal landmark whose inheritance can be studied Crime scene DNA Suspect DNA Fragment w Cut Fragment z Fragment x Cut Cut Fragment y Fragment y Figure Gel Electrophoresis The first step of RFLP analysis is to cut up a sample of DNA with a restriction enzyme This creates a mixture of restriction fragments The next step is to determine the number and size of fragments 13

14 Gel electrophoresis is a method for sorting these fragments based on their length and electrical charge Mixture of DNA fragments of different sizes Power source Gel Longer (slower) fragments Shorter (faster) fragments Completed gel Figure The DNA fragments are visualized as bands on the gel The bands of different DNA samples can then be compared Longer fragments Crime scene DNA Suspect DNA Shorter fragments Figure DNA fingerprints from a murder case Defendant s blood Blood on defendant s clothes Victim s blood Figure

15 GENOMICS Genomics is the science of studying whole genomes The first targets of genomics were pathogenic bacteria The Human Genome Project In 1990, an international consortium of governmentfunded researchers began the Human Genome Project The goal of the project was to sequence the human genome Figure Sequencing of the human genome presents a major challenge It is very large Only a small amount of our total DNA is contained in genes that code for proteins As of 2003, the genomes of over 100 organisms have been sequenced 15

16 Tracking the Anthrax Killer In October 2001, a Florida man died from inhalation anthrax By the end of the year, four other people had also died from anthrax Investigators analyzed the genome of the anthrax spores used in each attack Investigators were able to establish that the spores from all of the cases were identical This suggested a single perpetrator of the crime They were also able to match the anthrax with one laboratory subtype, the Ames strain This investigation is an example of the new field of comparative genomics 16

17 Genome-Mapping Techniques Human chromosomes range in size between 50 and 300 million base pairs Sequencing this much DNA at once is simply not possible Instead, chromosomes must be chopped up into smaller pieces Each piece is inserted into a vector and cloned The clone is then sequenced The public consortium divided the genome project into three stages Genetic (linkage) mapping Physical mapping DNA sequencing 1. Genetic (Linkage) Mapping Scientists combined pedigree analysis of large families with DNA technology to map over 5,000 genetic markers The resulting map provided anchor points These enabled researchers to easily map other markers by testing for genetic linkage to known markers 17

18 2. Physical Mapping Researchers used restriction enzymes to break the DNA of each chromosome into identifiable fragments These fragments were cloned Researchers then determined the original order of the fragments in the chromosome Genome mapping Chromosome Chop up with restriction enzyme DNA fragments Sequence fragments Align fragments Reassemble full sequence Figure DNA Sequencing The hardest part of the project was determining the nucleotide sequences of the set of DNA fragments created in stage 2 Automated sequencing machines worked around the clock These machines fed their results to computers that stored, analyzed, and reassembled the data Figure

19 The Whole Genome Shotgun Method In 1998, the private company Celera Genomics entered the race to map the human genome It was able to produce a draft of the human genome within three years How did they do this so quickly? The researchers at Celera pioneered a technique called the whole genome shotgun method This technique essentially skips the first two stages described and proceeds directly to the third In 2003, the international Human Genome Project concluded their sequencing work About 99% of the human genome was complete, leaving only a few regions unsequenced The DNA sequences determined by the public consortium are deposited in a database that is available on the Internet 19

20 HUMAN GENE THERAPY Human gene therapy is a recombinant DNA procedure that seeks to treat disease by altering the genes of the afflicted person The mutant version of a gene is replaced or supplemented with a properly functioning one Healthy individual 1 Normal human gene isolated and cloned Nonharmful virus (vector) 2 Human gene inserted into virus Virus containing normal human gene Bone marrow 3 Virus injected into patient Figure Treating Severe Combined Immunodeficiency SCID is a fatal inherited disease caused by a single defective gene The gene prevents the development of the immune system SCID patients quickly die unless treated with a bone marrow transplant 20

21 Human gene therapy has been used to treat people suffering from SCID In 2000, two infants suffering from SCID were provided with functional copies of their defective genes However, the SCID study was halted in 2002, after two patients developed leukemia-like symptoms SAFETY AND ETHICAL ISSUES As soon as scientists realized the power of DNA technology, they began to worry about potential dangers The creation of hazardous new pathogens The transfer of cancer genes into infectious bacteria and viruses Strict laboratory safety procedures have been designed to protect researchers from infection by engineered microbes Procedures have also been designed to prevent microbes from accidentally leaving the laboratory Figure

22 The Controversy Over Genetically Modified Foods GM strains account for a significant percentage of several agricultural crops in the United States In 1999, controversy over the safety of these foods prompted protests throughout Europe Figure Advocates of a cautious approach have two primary concerns Fear that crops carrying genes from other species might harm the environment Fear that GM foods could be hazardous to human health Negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol The protocol requires exporters to identify GM organisms present in bulk food shipments Several U.S. regulatory agencies evaluate biotechnology projects for potential risks Department of Agriculture Food and Drug Administration Environmental Protection Agency National Institutes of Health 22

23 Ethical Questions Raised by DNA Technology Should genetically engineered human growth hormone be used to stimulate growth in HGHdeficient children? Figure Genetic engineering of gametes and zygotes has been accomplished in lab animals Should we try to eliminate genetic defects in our children? Should we interfere with evolution in this way? Advances in genetic fingerprinting raise privacy issues What about the information obtained in the Human Genome Project? How do we prevent genetic information from being used in a discriminatory manner? EVOLUTION CONNECTION: GENOMES HOLD CLUES TO EVOLUTION DNA sequencing has confirmed evolutionary connections Such as between yeast cells and human cells Comparisons of DNA sequences strongly support the theory that there are three fundamental domains of life Bacteria Archaea Eukaryotes 23

24 SUMMARY OF KEY CONCEPTS Recombinant DNA Techniques Plasmid (vector) Recombinant DNA DNA isolated from two sources and cut by same restriction enzyme Gene of interest (could be synthesized or obtained from library) Transgenic organisms Visual Summary 12.1 Useful products DNA Fingerprinting Techniques Crime scene Suspect 1 Suspect 2 DNA DNA DNA Polymerase chain reaction (PCR) amplifies DNA samples Longer fragments Gel Visual Summary 12.2 Shorter fragments Human Gene Therapy Normal human gene Virus Bone marrow Visual Summary 12.3 Normal human gene is transcribed and translated in patient, potentially curing genetic disease permanently 24